Cavalcante et al. BMC Medical Genetics(2020) 21:207
https://doi.org/10.1186/s12881-020-01138-3

RESEARCH ARTICLE

Open Access

Investigation of INDEL variants in apoptosis:
the relevance to gastric cancer
Giovanna Chaves Cavalcante1, Milene Raiol de Moraes1, Cristina Maria Duarte Valente1, Caio Santos Silva1,
Antônio André Conde Modesto2, Paula Baraúna de Assumpção2, Paulo Pimentel de Assumpção2,
Sidney Santos1,2 and Ândrea Ribeiro-dos-Santos1,2*

Abstract
Background: Apoptosis is a type of cell death involved in different pathways inherent to the cell and the evasion
from this mechanism has been related to cancer, although this process remains not very well comprehended.
Gastric cancer (GC) is one of the most incident and aggressive types of cancer worldwide. In this study, we
analyzed the distribution of INDEL variants in GC patients (Case) and individuals from the general population(Control) from the Amazon region, in which GC is remarkably frequent.
Methods: A panel of nine INDEL markers in apoptosis-related genes (BCL2 rs11269260, CASP3 rs4647655, CASP8
rs3834129 and rs59308963, CASP9 rs4645982 and rs61079693, FADD rs4197, FAS rs10562972 and TP53 rs17880560)
was developed and genotyped by multiplex PCR in both groups.
Results: In our analyses, only marker rs4197 (FADD gene) was associated to GC development as follows: INS/DEL
genotype of rs4197 increasing in about 2-fold the chances of developing this type of cancer (P = 0.046; OR = 1.940;
95%CI = 1.011–3.725).
Conclusion: Our results suggest that rs4197 (FADD gene) might play a role in gastric carcinogenesis in theinvestigated population. More studies are needed to clarify this relation. Here, we highlight the importance ofinvestigating INDEL variants in genes involved in apoptosis.
Keywords: Gastric Cancer, Apoptosis, INDEL, FADD

Background
Apoptosis is a type of programmed cell death (PCD) that
may occur as a response to intra- or extracellular stimuli, leading to intrinsic pathway or extrinsic pathway, respectively. These are independent pathways thatconverge to the executor phase, the final stage of thisdeath process [1–3]. Several genes are involved in apoptosis, out of which we highlight: BCL2, CASP9 in the* Correspondence: akelyufpa@gmail.com
1
Laboratório de Genética Humana e Médica, Programa de Pós-Graduação
em Genética e Biologia Molecular, Universidade Federal do Pará, Av. Augusto
Correa 01, Belém, Pará 66075-110, Brazil
2
Núcleo de Pesquisas em Oncologia, Programa de Pós-Graduação em
Oncologia e Ciências Médicas, Universidade Federal do Pará, Belém, Pará
66073-005, Brazil

intrinsic pathway; FAS, FADD, CASP8 in the extrinsic
pathway; TP53 in both pathways; and CASP3 in the executor phase. All of these genes are important to theapoptotic mechanism, so that variants affecting theirfunctions might dysregulate cell homeostasis and contribute to tumor development [4]. Although the apoptosis process is still not completely comprehended, thistype of cell death mechanism has been related to cancer.
Gastric cancer (GC) is one of the most frequent andaggressive types of cancer worldwide, usually presenting
an unfavorable prognosis [5, 6]. In Brazil, GC is the sixth
most incident and the fifth most mortal type of cancer[6]. Brazil is a country with a highly admixed population,
composed mainly of European, African, Amerindian and© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Cavalcante et al. BMC Medical Genetics(2020) 21:207

Asian ancestry. Many studies in this country havehighlighted differences between these ancestries and theBrazilian regions [7–11], including aspects of cancer development [12]. This is also notable by the higher incidence of GC in northern Brazil in comparison to thegeneral rates of this country and the world [13].
Therefore, in this study, we aimed to analyze the distribution of nine Insertion/Deletion (INDEL) polymorphisms in apoptosis-related genes in gastric cancer
patients and cancer-free individuals from the general
population of the Amazon region.

Methods
Sampling

Case group was composed of 93 individuals with gastriccancer diagnosis from Pará state and control group wascomposed of 98 cancer-free individuals of the general
population from the same state, located in the Amazon
region of Brazil. From each individual, blood or buccal
swab samples were collected. All participants signed an
informed consent, with approval by the Committee for
Research Ethics of Hospital João de Barros Barreto
under Protocol Number: 64399617.7.0000.5634.
DNA extraction and quantification

DNA extraction was performed with phenol-chloroform
method, based on [14]. Quantification of DNA was performed with NanoDrop 1000 and Qubit™ (Thermo
Fisher Scientific, Wilmington, DE, USA).
Selection of INDEL markers

The markers were selected according to the main criteria: (i) they must be in genes involved in apoptosis

Page 2 of 6

pathways; (ii) they must be INDEL-type polymorphisms,
hence presenting the potential to modify the function of
the protein; (iii) they must have minor allele frequency(MAF) ≥ 10%. Table 1 shows the technical features of
the investigated markers.
Genotyping

The panel of markers described above was genotyped byMultiplex PCR, allowing the amplification of all of thesemarkers in a single reaction, followed by capillary electrophoresis and fragment analysis. Protocol preparationof PCR samples was: 5.0 μL of QIAGEN Multiplex PCR
Master Mix, 1.0 μL of Q-solution, 2.0 μL of water, 1.0 μL
of primer mix and 1.0 μL of DNA per sample. Amplification reaction was done using Veriti thermal cycler(Thermo Fisher Scientific) with the following protocol:
95 °C for 15 min, followed by 35 cycles of 94 °C for 45 s,
60 °C for 90 s and 72 °C for 1 min, with a final extension
at 70 °C for 30 min. The protocol for capillary electrophoresis was: 1.0 μL of PCR product, 8.5 μL of HI-DI deionized formamide and 0.5 μL of GeneScan 500 LIZ
pattern size standard (reagents by Thermo Fisher Scientific). DNA fragments were separated and analyzed using
ABI PRISM 3130 genetic analyzer and GeneMapper ID
v.3.2 software (both by Thermo Fisher Scientific).
In addition to the panel of apoptosis markers, a panelof 61 ancestry informative markers (AIM) previously developed and expanded by our research group was also
employed in this study, following the established protocols [15, 16]. This was done considering that the Brazilian population is highly admixed in terms of genetic
contributions from different parental populations,mainly European, African and Native American. In order

Table 1 Technical characterization of the markers included in the panel
Gene

ID

RegionAlleles

MAF

Primers

Amplicon

BCL2

rs11269260

IntronTCTATCACCGATCATT/−

0.37

F5’GCTTCCAGTTCCATCCATGT3’R5’CTCAGCGTGGTAGTGTTGGA3’

189–205

CASP3

rs4647655

Intron−/AAATCCTGAA

0.28

F5’AGGAGTATCCCCTCGTGGAC3’R5’CAAGAGTCAGGCAAAAACAGG3’

379–389

CASP8

rs3834129

Promoter

AGTAAG/−

0.39

F5’CTCTTCAATGCTTCCTTGAGGT3’
R5’CTGCATGCCAGGAGCTAAGTAT3’

249–255

CASP8

rs59308963

IntronATTCTGTC/−

0.26

F5’TTTTTGTCCTCCAAGCTTCC3’R5’GAACAAGAGAGAGGGCAGGA3’

261–269

CASP9

rs4645982

Intron−/TCCCCGCACTGACCTCACG

0.42

F5’GGTGACCCCAGAATTGACCCT3’R5’GCCCTCAGGACGCACCTCTG3’

336–353

CASP9

rs61079693

IntronAAAA/−

0.32

F5’CATGCACAGCTATCCAGGAG3’R5’TTGTTCCTGTCCGATAGATGC3’

458–462

FADD

rs4197

3′ UTR

−/TGT

0.47

F5’TGCCCCTACTTAGCAGTCTCA3’R5’GAGAGGTGGAGAACTGGGATT3’

278–281FAS

rs10562972

Intron

TTC/−

0.12

F5’GCATCAGGACGCTGAACATA3’R5’AATGCAACTTGCTCCAGAGG3’

368–371

TP53

rs17880560

3′-Flanking−/GCCGTG

0.21

F5’CTGTGTGTCTGAGGGGTGAA3’R5’ATCCTGCCACTTTCTGATGG3’

400–406

Cavalcante et al. BMC Medical Genetics(2020) 21:207

Page 3 of 6

to control the possible influence of these ancestries in
the genotypic distribution, leading to misinterpretation,
we have used this AIM panel, composed of ancestryspecific markers that are able to estimate individual andpopulation genetic contribution.

Nevertheless, in the analysis performed for each genotype (carriers of a genotype vs. non-carriers of suchgenotype as a reference group), only INS/DEL genotypeof rs4197 (P = 0.046; OR = 1.940; 95%CI = 1.011–3.725)
presented significant association to risk of developing
GC in the investigated population (Table 3).

Statistical analysis

Inference of genetic ancestry based on the AIM panel
was done with Structure software v.2.3.4 [17]. R language [18] was used to assess Hardy-Weinberg Equilibrium (HWE) of genotype distribution. JASP software v.
0.9.2.0 [19] was used to perform all other statistical analyses (Student’s t test, chi-squared test, logistic regression). P-values ≤0.05 were considered statisticallysignificant.

Results
Firstly, we assessed HWE for genotypic distribution andfound that only two markers, rs4645982 (CASP9) andrs4197 (FADD), deviated from HWE in case group (P =0.0035 and P = 0.0026, respectively).
Then, we compared sex, age and controlled genetic
ancestry of case and control groups in order to verify
possible confounding factors. In addition, staging (I-IV)of patients was assessed. These data are described inTable 2.
In these analyses, only age presented a statistically significant difference and it was included for correction in
the panel analysis. It is noteworthy that 53% of the patients included in case group presented diffuse-typeadenocarcinoma, while 47% presented intestinal-typeadenocarcinoma, and the observed mean age (meanage ± SE) for these subtypes was 39.20 ± 1.397 and44.34 ± 1.526, respectively, so that the mean age for casegroup as a whole was lower than it is usually observedfor patients with GC in the world.
Table 2 Demographic data for case (gastric cancer patients)
and control groups
P-value

Variable

Case

ControlN

91

95

Age, yearsa

42.15 ± 1.66

32.18 ± 1.17

< 0.001

Sex, % of male/femaleb

48.9/51.1

50.0/50.0

1.000European ancestryc

0.541 ± 0.028

0.587 ± 0.021

0.251

African ancestryc

0.152 ± 0.018

0.124 ± 0.013

0.966

Native American ancestry

0.307 ± 0.022

0.289 ± 0.019

0.649

Stage I, %

3.3

NA

0.241

Stage II, %

20.7

NA

Stage III, %

26.0

NA

Stage IV, %

50.0

NA

c

a

Values are expressed as mean ± SE (Standard Error of Mean), Student’s t-test;
b cValues are expressed as distribution percentages, chi-squared test; cValues
are expressed as mean ± SE, Mann-Whitney test. NA Not Applicable

Discussion
In the last decade, it has been established that tumors
display different abilities in order to grow, survive andproliferate, of which we highlight here resistance to cell
death, including apoptosis [20]. However, there is still alot to discover about these mechanisms.
Apoptosis is a complex process that involves the protein
action of several different genes, such as BCL2, CASP3,
CASP8, CASP9, FADD, FAS and TP53. Dysfunction inthese genes may lead to deregulation of cell death and, thus,
tumor development. Therefore, in this study, we investigated whether nine INDEL variants in the mentioned genes
might influence gastric carcinogenesis, by comparing theirdistribution in GC patients and cancer-free individuals.
Regarding the observed deviation from HWE in the
distribution of rs4197 and rs4645982 in case group, noprevious studies were found with these markers, but
their genotype distribution varies greatly between different populations in 1000 Genomes Project database [21].
Curiously, their distribution also presented a deviationfrom HWE in most populations in that database, suggesting that there could be a selective advantage leading
to this pattern and/or that the deviation observed here
could be due to population substructure, especially considering the relatively recent admixture process in Brazil,
as previously observed for other markers in this population [7, 22]. As such, it is an expected process inadmixed populations, and it could even highlight the potential of these markers. In fact, it has been suggested
that HWE deviation only in patient’s group could support possible locus-disease associations [23].
Then, we carried on to the analyses performed foreach genotype (carriers of a genotype vs. non-carriers ofsuch genotype as a reference group) in cases and controls. In these analyses, our results suggest that individuals carrying INS/DEL of rs4197 have about 2-foldchances of developing GC than non-carriers (P = 0.046;
OR = 1.940; 95%CI = 1.011–3.725).
To the best of our knowledge, this is the first study investigating variant rs4197. It is a 3-bp INDEL in the 3′
UTR of FADD gene, a key adaptor molecule that transmits death signals from death receptors during extrinsic
apoptosis, thus being crucial to a variety of processes[24]. In fact, it seems that it could affect protein featuresdue to the importance of 3′-UTR [25]. Depletion of
FADD protein action may lead to failure of apoptosis
and, thus, to tumor development. Absence of FADD

Cavalcante et al. BMC Medical Genetics(2020) 21:207

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Table 3 Genotypic distribution of the investigated variants for patients with GC in comparison to control group. P-value, OR and
95%CI were obtained with logistic regression adjusted for age, which was done for the genotype as reference in each line (DEL/DEL
vs. the other genotypes of that marker, and the same for INS/DEL and INS/INS)
Gene

Genotype

Case (%)*

Control (%)*

BCL2

rs11269260

93

98

CASP3

CASP8

CASP9

FADD

FAS

TP53

P-value

OR (95%CI)

DEL/DEL

13 (14)19 (19.4)

0.844

1.090 (0.463–2.567)

INS/DEL51 (54.8)

42 (42.9)

0.149

1.589 (0.848–2.977)

INS/INS

29 (31.2)

37 (37.8)

0.092

0.556 (0.281–1.101)

rs4647655

93

96

DEL/DEL47 (50.5)

48 (50.0)

0.898

0.959 (0.511–1.802)

INS/DEL39 (41.9)

39 (40.6)

0.995

1.002 (0.528–1.904)

0.825

1.136 (0.365–3.537)

INS/INS

7 (7.5)9 (9.4)

rs3834129

93

98

DEL/DEL27 (29.0)

19 (19.4)

0.098

1.851 (0.893–3.835)

INS/DEL38 (40.9)42 (42.9)0.987

0.995 (0.526–1.880)

INS/INS

28 (30.1)

37 (37.7)

0.132

0.596 (0.303–1.169)

rs59308963

89

93

DEL/DEL30 (33.7)

29 (31.2)

0.749

1.116 (0.568–2.195)

INS/DEL41 (46.1)

44 (47.3)

0.727

0.893 (0.471–1.690)

INS/INS

18 (20.2)

20 (21.5)

0.953

1.024 (0.467–2.244)

rs4645982

81

58

DEL/DEL

16 (19.8)

13 (22.4)

0.897

0.943 (0.392–2.272)

INS/DEL24 (29.6)

20 (34.5)

0.583

0.808 (0.377–1.732)

INS/INS

41 (50.6)

25 (43.1)

0.535

1.254 (0.613–2.567)

rs61079693

89

94

DEL/DEL

22 (24.7)

26 (27.7)

0.418

0.742 (0.360–1.529)

INS/DEL46 (51.7)

44 (46.8)

0.765

1.102 (0.585–2.076)

INS/INS

21 (23.6)

24 (25.5)

0.621

1.209 (0.570–2.563)

rs419793

97

DEL/DEL31 (33.3)

41 (42.3)

0.099

0.573 (0.295–1.111)

INS/DEL57 (61.3)

48 (49.5)

0.046

1.940 (1.011–3.725)

0.441

0.600 (0.163–2.202)

INS/INS

5 (5.4)8 (8.2)

rs10562972

89

93

DEL/DEL

1 (1.1)0 (0.0)0.988

991,452.554 (0.000–∞)

INS/DEL

10 (11.2)

21 (22.6)

0.260

0.603 (0.250–1.454)

INS/INS78 (87.6)

72 (77.4)

0.329

1.535 (0.650–1.125)

rs17880560

87

93

DEL/DEL57 (65.5)

58 (62.4)

0.738

1.119 (0.579–2.166)

INS/DEL26 (29.9)31 (33.3)

0.714

0.880 (0.445–1.741)

INS/INS

4 (4.6)4 (4.3)0.964

1.035 (0.232–4.623)*Sample number; P-value: P-value adjusted for age, OR Odds Ratio, CI Confidence Interval

expression has been suggested as marker of tumor development in mice and cancer prognosis in humans, due tothe involvement of this protein with cell apoptosis, survival and proliferation [26]. Therefore, investigation of
FADD gene should help the understanding of cancer development, including gastric carcinogenesis.

As for the variants that did not present a statistical association here, this may suggest that they either do notplay a role in gastric carcinogenesis or it is a populationspecific treat. Indeed, some of these variants have been
associated cancer development in a few studies, but notin others. For instance, one of these variants (rs3834129

Cavalcante et al. BMC Medical Genetics(2020) 21:207

in CASP8) has been previously studied by our researchgroup in other sampling of the same region: we have associated INS/INS genotype of this variant to reduced
chances of developing B-cell acute lymphoblastic
leukemia [27], but have not found any association of thisvariant with GC or colorectal cancer [22], so that ourstudy corroborates the findings in the latter. Thus, theassociation of such variants to GC and other types ofcancer is still a matter of great discussion.

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Ethics approval and consent to participate
All participants signed an informed consent, with approval by theCommittee for Research Ethics of Hospital João de Barros Barreto underProtocol Number: 64399617.7.0000.5634.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 27 February 2020 Accepted: 4 October 2020

Conclusions
Despite a few limitations (sample number and averageage of both groups), this study contributed to an increased knowledge on variants in apoptosis-related genes
in regard to GC development. As future perspectives, werecommend studies in the same population with agreater sample number and in different populations forcomparison, as well as functional studies focused on theproteins to possibly reinforce the involvement of FADD
gene in gastric carcinogenesis. Although such futurestudies are recommended to strengthen our results, thiswork contributes to a better understanding of thesegenes and INDEL variants in regard to gastric cancer.
Abbreviations
AIM: Ancestry Informative Marker; BCL2: BCL2 apoptosis regulator;
CASP3: Caspase 3; CASP8: Caspase 8; CASP9: Caspase 9; CI: ConfidenceInterval; DEL: Deletion; DNA: Deoxyribonucleic Acid; FADD: Fas associated via
death domain; FAS: Fas cell surface death receptor; GC: Gastric Cancer;
HWE: Hardy-Weinberg Equilibrium; ID: Identification; INDEL: Insertion/
Deletion; INS: Insertion; MAF: Minor Allele Frequency; OR: Odds Ratio;
PCD: Programmed Cell Death; PCR: Polymerase chain reaction; TP53: Tumor
protein p53; UTR: Untranslated Region
Acknowledgements
We thank Dr. André M. Ribeiro-dos-Santos for great technical assistance.
Authors’ contributions
GCC designed the study, performed part of the experiments, performed data
analysis and wrote the manuscript; MRM, CMDV, CSS and AACM performed
part of the experiments; PBA and PPA participated in the acquisition of dataand gave contributions to the manuscript; SS gave major contributions tothe study design and to the manuscript; ARS designed and coordinated thestudy and gave major contributions to the manuscript. The author(s) readand approved the final manuscript.
Funding
This research was funded by Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento dePessoal de Nível Superior (CAPES/Brazil), Fundação Amazônia de Amparo a
Estudos e Pesquisas (FAPESPA), Pró-Reitoria de Pesquisa e Pós-Graduação
(PROPESP/UFPA) and Fundação de Amparo e Desenvolvimento da Pesquisa(FADESP/UFPA). This work is part of Rede de Pesquisa em GenômicaPopulacional Humana (Biocomputacional – Protocol no. 3381/2013/CAPES).
The funders had no role in any of the following: design of the study;
collection, analyses, or interpretation of data; writing of the manuscript;
decision to publish the results.
Availability of data and materials
The datasets generated and/or analyzed during the current study areavailable in the Figshare repository (https://doi.org/10.6084/m9.figshare.
13040078).

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